AbstractThe composition of stable carbon and nitrogen isotopes (δ¹³C and δ¹⁵N) is widely used for tracking the origin of organic matter (OM) present in lacustrine sediments. These isotopes also define the evolution of OM in food web loops. Charophyte species Nitella flexilis C. Agardh, 1824 can be found in different aquatic environments where it contributes significantly to sediment formation and influences biota function. Therefore, it is crucial to study more about δ¹³C and δ¹⁵N in different lake types. Here, we present the results of the first comprehensive study of N. flexilis δ¹³C and δ¹⁵N, which add to the knowledge of the C and N isotope records of charophytes. We obtained the δ¹³C and δ¹⁵N records of N. flexilis OM from hardwater and softwater lakes and checked for differences between these records. We also analyzed the differences in physical and chemical parameters. Finally, we compared the δ¹³C and δ¹⁵N records with physical and chemical parameters to identify the variables that have the highest influence on N. flexilis δ¹³C and δ¹⁵N values. Our study showed that both δ¹³C and δ¹⁵N did not differ significantly in the two types of lakes, although the lakes had significant differences in several physical and chemical parameters (pH, Ca²⁺, dissolved inorganic carbon, total phosphorus, conductivity). However, we observed that δ¹³C values were influenced by light conditions (photosynthetic active radiation, depth, dissolved OM), while δ¹⁵N values were influenced by the total nitrogen concentration in water.